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Lookup NU author(s): Dr Hamed Mosaei Sejzi, Professor Nikolay ZenkinORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.
Author(s): Mosaei H, Zenkin N
Publication type: Article
Publication status: Published
Journal: Nucleic Acids Research
Year: 2021
Volume: 49
Issue: 15
Pages: 8777-8784
Print publication date: 07/09/2021
Online publication date: 07/08/2021
Acceptance date: 25/07/2021
Date deposited: 13/10/2021
ISSN (print): 0305-1048
ISSN (electronic): 1362-4962
Publisher: Oxford University Press
URL: https://doi.org/10.1093/nar/gkab675
DOI: 10.1093/nar/gkab675
PubMed id: 34365509
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